Propionic acids, propionic acid esters, and related compounds

ABSTRACT

The invention relates to compounds of the following formula: 
     
       
         
         
             
             
         
       
         
         
           
             and their pharmaceutically acceptable salts, pharmaceutical compositions containing this compounds, and methods of using this compound for the treatment of various diseases or conditions, including but not limited to diseases and/or conditions of, or involving, the Central Nervous System (CNS), including schizophrenia adrenoleukodystrophy, mitochondrial diseases (e.g. Leigh syndrome, Alpers&#39; disease, and MELAS), Huntington&#39;s disease, trichotillomania, HIV-associated neurocognitive disorder, hypoxic-ischemic encephalopathy, drug craving, and drug addiction.

FIELD OF THE INVENTION

This invention relates to novel propionic acids, propionic acid esters, and related compounds and methods of using these compounds for the treatment of diseases and/or conditions, including but not limited to diseases and/or conditions of, or involving, the Central Nervous System (CNS), including schizophrenia, adrenoleukodystrophy, mitochondrial diseases (e.g. Leigh syndrome, Alpers' disease, and MELAS), Huntington's disease, trichotillomania, HIV-associated neurocognitive disorder, hypoxic-ischemic encephalopathy, drug craving, and drug addiction.

BACKGROUND OF THE INVENTION

Schizophrenia is a debilitating disorder afflicting 1% of the world's population. The development of effective medications to treat schizophrenia relies on advances in characterizing the underlying pathophysiology. Chlorpromazine and other phenothiazines are considered first generation antipsychotics (termed “typical antipsychotics”) useful in the treatment of schizophrenia.

Schizophrenia may be associated with abnormal glutamate signaling and diminished glutathione levels. Impaired cystine-glutamate antiporter activity can lead to increased oxidative stress and depleted glutathione, as well as abnormal glutamate neurotransmission, synaptic connection, and gene expression, all of which are observed in schizophrenia. In addition, impaired cystine-glutamate antiporter activity and faulty glutamate neurotransmission bear on the issue of uncontrolled drug use, i.e., drug addiction.

Adrenoleukodystrophy, or X-linked adrenoleukodystrophy, is an inherited life-threatening metabolic rare disease. It primarily affects myelination throughout the nervous system, the adrenal cortex, and the Leydig cells in the testes where very-long chain fatty acids accumulate. The adrenoleukodystrophy patient population is heterogeneous, with clinical phenotypes that include progressive neurodegenerative decline leading to vegetative state in children (childhood cerebral X-linked adrenoleukodystrophy). Treatments for adrenoleukodystrophy are include hematopoietic stem cell transplant, which results in a high-survival rate (92% 5-year survival; Peters et al. “Cerebral X-linked adrenoleukodystrophy: the international hematopoietic cell transplantation experience from 1982 to 1999,” Blood. 2004; 104:881-888.); however, this treatment is limited to and effective with only a small adrenoleukodystrophy subpopulation, with success typically corning when it is performed in the early stages of the disease.

Adrenoleukodystrophy patients have one or more mutations to the ABCD1 gene, which encodes the peroxisomal ATP-binding cassette transporter. Subsequently, very-long chain fatty acids build up in affected cells, leading to oxidative stress and eventually metabolic failure resulting in cell death, features common to human patients and animal models (Fourcade, S, et al. “Early oxidative damage in neurodegeneration Underlying X-adrenoleukodystrophy.” Human Molecular Genetics, 2008; 17: 1762-1773; López-Erauskin, J et al. “Antioxidants halt axonal degeneration in a mouse model of X-adrenoleukodystrophy.” Annals of Neurology, 2011; 70:84-92).

Antioxidant therapy is a promising therapy for the treatment of adrenoleukodystrophy and other diseases that involve oxidative stress. At the cellular level, antioxidants have been demonstrated to normalize biomarkers of oxidative stress. N-acetylcysteine (“NAC”) is a prodrug of cysteine, which serves as the limiting reagent in the synthesis of glutathione, the body's major antioxidant. When given as an adjuvant therapy to hematopoietic stem cell transplant in advanced stage childhood cerebral X-linked adrenoleukodystrophy patients, patient survival outcome greatly improves with NAC treatment (Miller, W et al. “Outcomes after allogeneic hematopoietic cell transplantation for childhood cerebral adrenoleukodystrophy: the largest single-institution cohort report, 2011; 118:1971-1978: Tolar, J et al. “N-acetyl-L-cysteine improves outcome of advanced cerebral adrenoleukodystrophy.” Bone Marrow Transplant, 2007; 39:211-215). However, brain penetrance is low and the long-term risks and benefits remain unknown.

Inherited mitochondrial diseases (e.g. Leigh syndrome, Alpers' disease, and MELAS) affecting the CNS are highly variable, and often result in the progressive loss, or dysfunction, of neurons or neuroglial cells. In many cases, the pathogenesis is a result of disruption of mitochondrial respiratory chain processes, which can then increase the generation of reactive oxidative species (“ROS”), due to mutations in mitochondrial or nuclear DNA. Antioxidant therapy, specifically N-acetylcysteine, acts to decrease ROS and increases glutathione levels, which concomitantly increase cell survival and function.

A range of other diseases share common pathophysiology with abnormal glutamate signaling and heightened levels of oxidative stress, particularly with System x_(c)-, a glutamate-cystine antiporter. Therefore, by engaging a single target, e.g. System x_(c)-, which is at the junction of two distinct metabolic pathways, NAC, NAC derivatives and related molecules, may effectively treat these wide-ranging, and seemingly unrelated, diseases and disorders. This has been partially demonstrated in clinical study with NAC treatment of trichotillomania (Grant, J E, et al, “N-Acetylcysteine, a Glutamate Modulator, in the Treatment of Trichotillomania.” Arch Gen Psychiatry, 2009: 66: 756-763). System x_(c)-, NAC, and disturbances in glutamate signaling and oxidative stress are also linked to other diseases that include, but are not limited to, Huntington's disease (Frederick, N M, et al. “Dysregulation of system xc(-) expression induced by mutant huntingtin in a striatal neuronal cell line and in R6/2 mice.” Neurochem Int, 2014; 76: 59-69), hypoxic-ischemic encephalopathy (Wang, X, et al. “N-acetylcysteine reduces lipopolysaccharide-sensitized hypoxic-ischemic brain injury.” Ann Neurol, 2007: 61: 263-271), HIV-associated neurocognitive disorder (Vázquez-Santiago F J, et al. “Glutamate metabolism and HIV-associated neurocognitive disorders.” J Neurovirol, 2014; 20: 315-331).

Cysteine prodrugs, such as NAC, drive cystine-glutamate exchange by apparently elevating extracellular cystine levels, thereby creating a steep cystine concentration gradient.

However, alternatives to NAC are needed. NAC undergoes extensive first pass metabolism requiring the usage of high doses that limit the utility of the drug and, potentially, increase the chances of side effects due to the buildup of metabolized by-products. The compounds of the present invention are designed to substantially avoid the problem of first pass metabolism and therefore exhibit increased efficacy as compared to NAC and other prior cysteine prodrugs.

Accordingly, there is a need for novel compounds that would have a reduced incidence of problems associated with NAC.

SUMMARY OF THE INVENTION

In one embodiment, the present invention is directed to a compound of formula I

wherein R¹ and R² are selected from the group consisting of H and a linear, branched or cyclic C₁-C₆ alkyl; or a pharmaceutically acceptable salt thereof

In another embodiment, the present invention is directed to a compound selected from a group consisting of:

In a preferred embodiment, the present invention is directed to a compound of the following formula:

This compound can be named (R)-ethyl-2-acetamido-3-(((S)-2-amino-3-methylbutanoyl)thio)propanoate.

The invention also encompasses pharmaceutically acceptable salts of this compound, including but not limited to a hydrochloride salt:

This compound is also referred to as “Pro-4001” throughout the specification.

In another aspect, the invention is directed to a method of treating a disease or condition in a subject comprising administering to the subject a therapeutically effective amount of one or more compounds of the invention or a pharmaceutically acceptable salt thereof. The preferred route of administering to the subject is via oral delivery. Preferably, diseases or conditions treatable with the compounds of the present invention are related to central nervous system (CNS).

In a preferred embodiment, the disease is schizophrenia.

In another aspect, the invention provides a method of treating drug craving in a subject comprising administering to the subject a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof. The preferred route of administering to the subject is via oral delivery.

The invention further encompasses pharmaceutical compositions containing the compound of the invention or a pharmaceutically acceptable salt thereof in combination with a pharmaceutically-acceptable carrier.

Methods of formulating/manufacturing such pharmaceutical compositions (alternatively termed “medicaments”) for the treatment of a disease or condition in a subject are also within the invention's scope.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of the ¹⁴C-cystine uptake as a percentage of the control.

FIG. 2 is a graphical representation of the ³H-glutamate release as a percentage of the control.

FIG. 3 is a graphical representation of the amount of intracellular cysteine.

FIG. 4 is a graphical representation of prepulse inhibition for Pro-4001.

FIG. 5 is a graphical representation of the total time Pro-4001 treated rats spent on the open arms of the elevated-plus maze.

* denotes a statistically significant difference from control.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are used, unless otherwise described.

The term “subject” includes humans. The terms “human,” “patient” and “subject” are used interchangeably.

The term “therapeutically effective amount” means the amount of a compound that, when administered to a subject for treating a disease or disorder, is sufficient to effect such treatment for the disease or disorder. The “therapeutically effective amount” can vary depending on the compound, the disease or disorder and its severity, and the age, weight, etc., of the subject to be treated.

The terms “treating” or “treatment” of any disease or disorder refer, in one embodiment, to ameliorating the disease or disorder (i.e., arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment “treating” or “treatment” refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, “treating” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In yet another embodiment, “treating” or “treatment” refers to delaying the onset of the disease or disorder, or even preventing the same.

The term “prodrugs” refers to compounds, including monomers and dimers of the compounds of the invention, which have cleavable groups and become under physiological conditions compounds which are pharmaceutically active in vivo.

The term alkyl as used herein indicates a linear, branched or cyclic alkyl groups having 1 to 6 carbon atoms. They include, for example, methyl group, ethyl group, n-propyl group, n-butyl group, n-pentyl group, n-hexyl group, isopropyl group, isobutyl group, sec-butyl group, tert-butyl group, isopentyl group, tertpentyl group, neopentyl group, 2-pentyl group, 3-pentyl group, 3-hexyl group, 2-hexyl group, cyclopropyl group, cyclobutyl group, cyclopentyl group and cyclohexyl group.

In one embodiment, the present invention is directed to a compound of formula I

wherein R¹ and R² are selected from the group consisting of H and a linear, branched or cyclic C₁-C₆ alkyl; or a pharmaceutically acceptable salt thereof

In another embodiment, the present invention is directed to a compound selected from a group consisting of:

In a preferred embodiment, the present invention is directed to a compound of the following formula:

The invention also encompasses pharmaceutically acceptable salts of this compound, including but not limited to a hydrochloride salt:

The compounds of the invention may potentially exist in different isomeric (e.g. enantiomers and distereoisomers) forms. The invention contemplates all such isomers both in pure form and in admixture, including racemic mixtures. Enol forms are also included.

The compounds of the invention can exist in unsolvated as well as solvated forms, including hydrated forms, e.g., hemi-hydrate. In general, the solvated forms, with pharmaceutically acceptable solvents such as water, ethanol, and the like are equivalent to the unsolvated forms for the purposes of the invention.

The compounds of the invention may also form pharmaceutically acceptable salts, e.g., acid addition salts. For example, the nitrogen atoms may form salts with acids. Examples of suitable acids for salt formation are hydrochloric, sulfuric, phosphoric, acetic, citric, oxalic, malonic, salicylic, malic, furmaric, succinic, ascorbic, maleic, methanesulfonic and other mineral carboxylic acids well known to those in the art. The salts are prepared by contacting the free base form with a sufficient amount of the desired acid to produce a salt in the conventional manner. The free base forms may be regenerated by treating the salt with a suitable dilute aqueous base solution such as dilute aqueous hydroxide potassium carbonate, ammonia, and sodium bicarbonate. The free base forms differ from their respective salt forms somewhat in certain physical properties, such as solubility in polar solvents, but the acid salts are equivalent to their respective free base forms for purposes of the invention. (See, for example S. M. Berge, et al., “Pharmaceutical Salts,” J. Pharm. Sci., 66: 1-19 (1977) which is incorporated herein by reference.)

As used herein, the term “composition” is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from a combination of the specified ingredients in the specified amounts.

The compounds of the present invention can be used in the form of pharmaceutically acceptable salts derived from inorganic or organic acids. The phrase “pharmaceutically acceptable salt” means those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well-known in the art. For example, S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1 et seq. The salts can be prepared in situ during the final isolation and purification of the compounds of the invention or separately by reacting a free base function with a suitable organic acid. Representative acid addition salts include, but are not limited to acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethansulfonate (isothionate), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmitoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate and undecanoate. Also, the basic nitrogen-containing groups can be quaternized with such agents as lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; arylalkyl halides like benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained. Examples of acids which can be employed to form pharmaceutically acceptable acid addition salts include such inorganic acids as hydrochloric acid, hydrobromic acid, sulphuric acid and phosphoric acid and such organic acids as oxalic acid, maleic acid, succinic acid and citric acid.

Basic addition salts can be prepared in situ during the final isolation and purification of compounds of this invention by reacting a carboxylic acid-containing moiety with a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia or an organic primary, secondary or tertiary amine. Pharmaceutically acceptable salts include, but are not limited to, cations based on alkali metals or alkaline earth metals such as lithium, sodium, potassium, calcium, magnesium and aluminum salts and the like and nontoxic quaternary ammonia and amine cations including ammonium, tetramethylammonium, tetraethylammonium, methylammonium, dimethylammonium, trimethylammonium, triethylammonium, diethylammonium, and ethylammonium among others. Other representative organic amines useful for the formation of base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine and the like.

Dosage forms for topical administration of the compounds of this invention include powders, sprays, ointments and inhalants. The active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives, buffers or propellants which can be required. Ophthalmic formulations, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.

Actual dosage levels of active ingredients in the pharmaceutical compositions of this invention can be varied so as to obtain an amount of the active compound which is effective to achieve the desired therapeutic response for a particular patient, compositions and mode of administration. The selected dosage level will depend upon the activity of the particular compound, the route of administration, the severity of the condition being treated and the condition and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

When used in the above or other treatments, a therapeutically effective amount of the compounds of the present invention can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt, ester or prodrug form. Alternatively, the compound can be administered as a pharmaceutical composition containing the compound of interest in combination with one or more pharmaceutically acceptable excipients.

The phrase “therapeutically effective amount” of the compounds of the invention means a sufficient amount of the compound to treat disorders, at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

The total daily dose of the compounds of this invention administered to a human or lower animal may range from about 0.0001 to about 2000 mg/kg/day. For purposes of oral administration, more preferable doses can be in the range of from about 0.001 to about 15 mg/kg/day, with the most preferable dose being in the range of from about 0.001 to about 5 mg/kg/day. If desired, the effective daily dose can be divided into multiple doses for purposes of administration; consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose.

The present invention also provides pharmaceutical compositions that comprise compounds of the present invention formulated together with one or more non-toxic pharmaceutically acceptable carriers. The pharmaceutical compositions can be specially formulated for oral administration in solid or liquid form, for parenteral injection or for rectal administration.

The pharmaceutical compositions of this invention can be administered to humans and other mammals orally, rectally, parenterally, intracisternally, intravaginally, transdermally (e.g. using a patch), transmucosally, sublingually, pulmonary, intraperitoneally, topically (as by powders, ointments or drops), bucally or as an oral or nasal spray. The term “parenterally,” as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

In another aspect, the present invention provides a pharmaceutical composition comprising compounds of the present invention and a physiologically tolerable diluent. The present invention includes one or more compounds as described above formulated into compositions together with one or more non-toxic physiologically tolerable or acceptable diluents, carriers, adjuvants or vehicles that are collectively referred to herein as diluents, for parenteral injection, for intranasal delivery, for oral administration in solid or liquid form, for rectal or topical administration, among others.

Compositions suitable for parenteral injection may comprise physiologically acceptable, sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), vegetable oils (such as olive oil), injectable organic esters such as ethyl oleate, and suitable mixtures thereof.

These compositions can also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

Suspensions, in addition to the active compounds, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.

Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound may be mixed with at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol and silicic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; c) humectants such as glycerol; d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) solution retarding agents such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) cetyl alcohol, glycerol monostearate, and PEG caprylic/capric glycerides; h) absorbents such as kaolin and bentonite clay and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

The solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells such as enteric coatings and other coatings well-known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also be of a composition such that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.

The active compound can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan and mixtures thereof.

Besides inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.

Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

Compounds of the present invention can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals which are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to a compound of the present invention, stabilizers, preservatives, excipients and the like. The preferred lipids are natural and synthetic phospholipids and phosphatidyl cholines (lecithins) used separately or together.

Methods to form liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq.

In another aspect, the invention is directed to a method of treating a disease or condition in a subject comprising administering to the subject a therapeutically effective amount of the compound of the invention or a pharmaceutically acceptable salt thereof. The preferred route of administering to the subject is via oral delivery.

Preferably, diseases or conditions treatable with the compounds of the present invention are related to central nervous system (CNS). In a preferred embodiment, the disease is schizophrenia.

However, it is within a skill in the art that the provided compound may be used to treat other diseases or conditions associated with diminished glutathione levels and/or glutamate signaling, and/or oxidative stress, and/or impaired cystine-glutamate antiporter activity, glutamate neurotransmission, synaptic connection, and gene expression.

In general, the invention is not limited to treatment of any specific disease or condition but encompasses the treatment of any disease or condition whose mechanism may be affected by the compounds of the present invention.

In another aspect, the invention provides a method of treating drug craving in a subject comprising administering to the subject a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof. The preferred route of administering to the subject is via oral delivery.

The invention further encompasses pharmaceutical compositions containing a compound of the invention or a pharmaceutically acceptable salt thereof in combination with a pharmaceutically-acceptable carrier.

Methods of formulating/manufacturing such pharmaceutical compositions (alternatively termed “medicaments”) for the treatment of a disease or condition in a subject are also within the invention's scope.

For a clearer understanding of the invention, details are provided below. These are merely illustrations and are not to be understood as limiting the scope of the invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the following examples and foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

EXAMPLES

Exemplary synthetic strategies of the compound of the invention are outlined below.

Example 1 Synthetic Strategies to Prepare Compounds of Formula (I)

Pathway A: L-cysteine, a known compound, is reacted with a suitably substituted carboxylic acid, of formula (II), a known compound or compound prepared by known methods and a coupling agent such as EDC, HOBt, PyBROP, DCC, and the like, and a base, such as triethylamine, diisopropylethylamine, N-methylmorpholine and the like, in an organic solvent like tetrahydrofuran, N,N-dimethylformamide, dichloromethane, and the like, to give the compound of formula (III).

Pathway B: L-cysteine, a known compound, is reacted with a suitably substituted acyl halide, of formula (IV), a known compound or compound prepared by known methods and a base such as triethylamine, diisopropylethylamine, N-methylmorpholine and the like in an organic solvent like dichloromethane, tetrahydrofuran, toluene and the like, to give the compound of formula (III).

Pathway C: L-cysteine, a known compound, is reacted with a suitably substituted acyl benzotriazole, of formula (V), a known compound or compound prepared by known methods and a base such as triethylamine, diisopropylethylamine, N-methylmorpholine and the like in a solvent like acetonitrile, tetrahydrofuran, water and the like, to give the compound of formula (III).

Pathway D: L-cysteine, a known compound, is reacted with a suitably substituted nitrile, of formula (V), a known compound or compound prepared by known methods and a base such as potassium carbonate, sodium carbonate, sodium hydroxide and the like in a solvent like acetonitrile, methanol, water and the like, to give the compound of formula (III).

Pathway E: A suitably substituted compound of formula (III) is treated with a suitably substituted alcohol, formula (VII), a known compound or compound prepared by known methods and an acid, such as thionyl chloride, phosphorus oxychloride and the like at or near the refluxing temperature of the alcohol to give a compound of formula (VIII).

Pathway F: A suitably substituted compound of formula (VIII) is treated with suitably protected L-valine of formula (IX) and a coupling agent such as EDC, HOBt, PyBROP, DCC, and the like, and a base, such as triethylamine, diisopropylethylamine, N-methylmorpholine and the like, in an organic solvent like tetrahydrofuran, N,N-dimethylformamide, dichloromethane, and the like, to give a protected form of compound of formula (I). The protecting group can be remove by treatment under suitable conditions such as with acid, such as hydrogen chloride, trifluoroacetic acid, and the like in organic solvent such as 1,4-dioxane, dichloromethane, and the like, or hydrogen in the presence of a catalyst such as palladium on activated carbon, platinum oxide and the like in an organic solvent such as ethyl acetate, methanol, ethanol or 3) base such as sodium hydroxide, potassium carbonate and the like in a solvent like water, methanol, tetrahydrofuran and the like to give the compound or a salt of the compound of formula (I).

Example 2 Synthesis of ethyl (2R)-3-{[(2S)-2-amino-3-methylbutanoyl]sulfanyl}-2-acetamido-propanoate hydrochloride (AMRI-1)

Ethyl (2R)-3-{[(2S)-2-{[(tert-butoxy)carbonyl]amino}-3-methylbutanoyl]sulfanyl}-2-acetamidopropanoate

To a solution of L-N-Boc-valine (767 mg, 3.53 mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (677 mg, 3.53 mmol) and hydroxybenzotriazole hydrate (477 mg, 3.53 mmol) in N,N-dimethylformamide (2.25 mL) was added 2-acetylamino-3-mercapto-propionic acid ethyl ester (450 mg, 2.35 mmol) and the resulting slurry was stirred at ambient temperature for 72 hours during which time a solution formed. The mixture was diluted with aqueous hydrochloric acid (0.2 M, 300 mL) and extracted with ethyl acetate (4×200 mL). The combined organic layers were washed with saturated aqueous sodium hydrogen carbonate (150 mL). The organic layer was dried over magnesium sulfate, filtered and concentrated at reduced pressure. This crude product was purified with silica gel (120 g) chromatography eluting with 30 to 100% ethyl acetate in hexane. The purified fractions were combined and concentrated at reduced pressure. The resulting solid was triturated with ethyl acetate/hexanes (1:1, 100 mL), filtered and dried to give the product as a white solid (580 mg, 63%). ¹H NMR (300 MHz, CDCl₃) δ=6.30 (d, J=7.3 Hz, 1H), 4.95 (d, J=8.2 Hz, 1H), 4.84 (td, J=5.0, 7.9 Hz, 1H), 4.23-4.14 (m, 3H), 3.48 (d, J=4.4 Hz, 1H), 3.45-3.32 (m, 2H), 2.29-2.15 (m, 1H), 2.03-1.99 (m, 3H), 1.29 (t, J=7.2 Hz, 3H), 0.99 (d, J=7.0 Hz, 3H), 0.89 (d, J=7.0 Hz, 3H).

Ethyl (2R)-3-{[(2S)-2-amino-3-methylbutanoyl]sulfanyl}-2-acetamidopropanoate hydrochloride

ethyl (2R)-3-{[(2S)-2-{[(tert-butoxy)carbonyl]amino}-3-methylbutanoyl]sulfanyl}-2-acetamidopropanoate (570 mg) was dissolved in a solution of hydrogen chloride (4N) in 1,4-dioxane (15 mL) and the reaction was stirred for 5 hours. The reaction mixture was concentrated at reduced pressure, triturated with ethyl acetate (15 mL) for 1 hour, filtered and dried to give the product as a white solid (430 mg, 90%). ¹H NMR (300 MHz, DMSO) δ=8.63-8.48 (m, 4H), 4.41 (dt, J=5.3, 8.4 Hz, 1H), 4.17-4.04 (m, 4H), 3.55-3.45 (m, 2H), 3.12 (dd, J=8.6, 13.6 Hz, 1H), 2.17 (dt, J=5.0, 6.9 Hz, 1H), 1.83 (s, 3H), 1.21-1.15 (m, 3H), 0.96 (d, J=7.0 Hz, 3H), 0.93 (d, J=7.0 Hz, 3H). MS (ESI) m/z 291 (M+1)′.

Example 3 In Vitro Studies

¹⁴C Uptake by Compound of the Invention

The goal of these experiments was to determine ¹⁴C-cystine uptake.

The experiments were conducted as follows.

The screening of the compound was performed using an in vitro culture system of human glial cells from brain astrocytoma (1321N1). Cells were plated on 24 well plates coated with poly-D-lysine and laminin and grown in a balanced salt solution supplemented with 5% heat inactivated horse serum, 5% fetal bovine serum, 2 mM glutamine and glucose (total 21 mM). Cultures were maintained in humidified 5% CO₂ incubators at 37° C. for 3-4 days before experiments were performed, at this time the cultures has formed a single confluent layer. For experiments, cultures are washed 3 times into a Na-free HEPES and HCO₃ ⁻ buffered balanced salt solution. After 1 hour, the test compounds are added. Following a three hour incubation, ¹⁴C-cystine (0.025 mCi/mL) was then added for 20 minutes. Following the ¹⁴C-cystine exposure, cultures were washed 3 times with ice cold HEPES buffered saline solution and dissolved in 250 μl sodium dodecyl sulfate (0.1%). An aliquot (200 μl) was removed and added to scintillation fluid for counting. Values were normalized to ¹⁴C-cystine uptake in untreated controls on the same experimental plate.

³H-Glutamate Release

This assay also uses the cell culture system of human glial cells from brain astrocytoma (1321N1) described above. Initially, cells are washed, and ³H-glutamate is added (PerkinElmer: 1 mCi/mL stock solution is diluted (30 μL+500 μL HBBSS) and 10 μL of diluted radiolabel is added to each well). Following a 1 hour incubation to load the cells with the labeled glutamate, the cells are washed again with HBBSS, and the drug is added. At 30, 90, and 180 minutes, 50 μL of extracellular media is sampled from each well and is measured using a Beckman LS 6500 scintillation counter.

Results of the ¹⁴C-cystine uptake and ³H-glutamate release by astrocytes treated with differing concentrations of the cysteine prodrug, 2-acetylamino-3-(2-amino-3-methyl-butyrylsulfanyl)-propionic acid ethyl ester (“Pro-4001”) are presented in FIGS. 1 and 2, respectively, as a percentage of the untreated control. The most effective concentration of Pro-4001 was 100 μM which caused 50% decrease in ¹⁴C-cystine uptake demonstrating inhibition of ¹⁴C-cystine uptake by a process not limited to an alternate substrate, a cystine/glutamate antiporter inhibitor, and an effective prodrug. The corresponding 175% increase in ³H-glutamate release at 3 hours is evidence that the ¹⁴C-cystine uptake is being inhibited by direct competition from Pro-4001.

In Vitro Thiol Experiments

To further resolve by what process the inhibition of ¹⁴C-cystine uptake is occurring intracellular cysteine levels were determined.

The experiment was conducted as follows.

Mixed cortical cell cultures containing glial and neuronal cells were prepared from fetal (15-16 day gestation) mice as previously described. Lobner D, Comparison of the LDH and MTT assays for quantifying cell death: validity for neuronal apoptosis?, J Neurosci Methods, 2000, Mar. 15, 96(2), 147-152). Dissociated cortical cells were plated on 24-well plates coated with poly-D-lysine and laminin in Eagles' Minimal Essential Medium (MEM, Earle's salts, supplied glutamine-free) supplemented with 5% heat-inactivated horse serum, 5% fetal bovine serum, 2 mM glutamine and glucose (total 21 mM). Cultures were maintained in humidified 5% CO₂ incubators at 37° C. Mice were handled in accordance with a protocol approved by our institutional animal care committee and in compliance with the Public Heath service Policy on Humane Care and Use of Laboratory Animals.

Mixed cortical cell cultures, 14 days in vitro (this allows for a confluent layer of astrocystes to form and the neurons to generate a complex network of axons and dendrites), were washed into bicarbonate buffered salt solution. After 1 hour, 3, 10, 30, 100 μM of Pro-4001 was added and the cells were incubated for 30 or 90 minutes, after which the cells were thoroughly washed and the collected in 250 μL of aqueous mobile phase (50 mM citric acid, 10 mM octanesulfonic acid, pH 2.8) was added, after 10 minutes at 37° C. the cells were scraped from the plates and transferred to 1.5 mL tubes for analysis.

Samples were then sonicated with a Fisher Scientific 60 Sonic Dismembrator. One fraction of this homogenate was analyzed with the Pierce BCA (bicinchoninic acid) method to determine protein concentration. The other fraction was filtered using 3 K molecular weight cutoff, polyethersulfone centrifugal protein filters, and analyzed for thiol content using high-performance liquid chromatography (ALF-115 column, 150×1.0 mm, 3 μm C18; mobile phase: 50 mM citric acid, 10 mM octanesulfonic acid, 2% acetonitrile, pH 2.8, 50 uL/min flow rate) with electrochemical detection (Decade II, Au working electrode, Flex Cell HyREF, 0.55V, Antec Leyden, Netherlands).

Results (FIG. 3) demonstrate that administration of Pro-4001 at 100 μM increases intracellular cysteine concentration to 0.78 μM (approximately 5 times the control value). This result demonstrates that Pro-4001 was effectively cleaved, yielding an increase in intracellular cysteine. Thus, based on the 3 in vitro experiments it is apparent that Pro-4001 behaves as an effective cysteine prodrug.

Example 4 In Vivo Studies

Prepulse Inhibition Experiment

The goal of this experiment was to demonstrate the efficacy of the test compounds in a predictive animal model of schizophrenia.

The experiment was conducted as follows.

Rats were placed on a platform in a sound attenuating chamber (10.875″×14″×19.5″; Hamilton Kinder, Calif.) that rested on a motion sensing plate. During all sessions, the background noise was held constant at 60 dB. A matching session was conducted to determine the magnitude of the average startle response for each rat. This session consisted of a five minute habituation period followed by 20 trials; 17 trials involved the presentation of a single auditory stimulus (pulse stimulus; 50 dB above the background noise) and three trials in which a pre-pulse stimulus (12 db above background) was presented 100 ms before the pulse auditory stimulus. Rats were then assigned into the various treatment groups so that the magnitude of the startle response was equivalent across all groups. Two days later, a testing session was conducted to assess sensorimotor gating. One hour prior to testing, rats received a prodrug (0-100 mg/kg, P.O.) and 55 minutes later acute MK-801 maleate (0.1 mg/kg, SC). The testing session consisted of a five minute habituation period, after which rats received 58 discrete trials; 26 trials during which the pulse stimulus (50 db above background) was presented alone, eight trials each in which the pulse stimulus was preceded by a prepulse stimulus (5, 10, or 15 db above background) and eight background trials with no pulse (No stimulus; background noise only). The first six pulse alone trials were not included in the average startle stimulus to achieve a relatively stable level of startle reactivity. All startle responses were normalized to vehicle control, and the percent of prepulse inhibition was determined as 100−(average prepulse startle response/average startle stimulus alone)*100.

FIG. 4 demonstrates the results of the experiment for Pro-4001.

FIG. 4 depicts the bar graph of the average prepulse inhibition from the experiment which was done in triplicate. As the bar graph in FIG. 4 demonstrates, at 3 mg/kg concentration, % prepulse inhibition was about 27%, as compared to about 18% for the control group that received both MK-801 and vehicle.

These results indicate that this compound ameliorates the schizophrenia-like MK-801-induced deficit in prepulse inhibition.

Elevated-Plus Maze

The goal of this experiment was to demonstrate the ability of the test compounds to penetrate the CNS.

The experiment was conducted as follows.

Rats were tested in a standard elevated plus maze; testing occurred in a dimly illuminated room using only two lights mounted over the maze. Animals were allowed to habituate to the room for at least one hour prior to treatment. One hour prior to testing, rats received a Pro-4001 (0-30 mg/kg, P.O.). For testing, the rat was placed in the elevated plus maze for five minutes, alternating the starting position between facing an open arm and facing a closed arm. The session was recorded and an observer blind to treatment recorded the number of explorations, entries and time spent in the open arm. Explorations were defined as the rat placing two feet into an open arm without fully entering said arm. Entries were defined as the rat placing all four feet in an open arm. Time of entry in the open arm was recorded from the time the rat placed four feet in the open arm until two of the rats' feet entered the open square.

After administration of Pro-4001, the rat increased the amount of time spend on the open arms of the maze, demonstrating a reduced anxiety and effective alleviation of symptoms associated with schizophrenia. Specifically, as seen in FIG. 5, rats treated with 10 mg/kg, PO (orally) of Pro-4001 spent around 21 seconds on the open arms and those treated with 30 mg/kg, PO (orally) spent around 25 seconds on the open arms. This represents an increase of time spent on the open arm of 50% and 64%, respectively. Thus, Pro-4001 demonstrates the ability to alleviate symptoms associated with schizophrenia in vivo. 

What is claimed is:
 1. A compound of formula I

wherein R¹ and R² are each independently selected from the group consisting of H and a linear, branched or cyclic C₁-C₆ alkyl; or a pharmaceutically acceptable salt thereof.
 2. The compound of claim 1, wherein the compound is selected from a group consisting of:


3. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier.
 4. A method of treating a disease selected from the group consisting of adrenoleukodystrophy, Leigh syndrome, Alpers' disease, MELAS, Huntington's disease, trichotillomania, HIV-associated neurocognitive disorder, hypoxic-ischemic encephalopathy and schizophrenia comprising administering to a subject in need thereof a therapeutically effective amount of the compound of claim
 1. 5. The method of claim 4 wherein the disease is schizophrenia.
 6. A compound having the following formula,


7. A pharmaceutical composition comprising the compound of claim 6 and a pharmaceutically acceptable carrier.
 8. A method of treating a disease selected from the group consisting of adrenoleukodystrophy, Leigh syndrome, Alpers' disease, MELAS, Huntington's disease, trichotillomania, HIV-associated neurocognitive disorder, hypoxic-ischemic encephalopathy and schizophrenia comprising administering to a subject in need thereof a therapeutically effective amount of the compound of claim
 6. 9. The method of claim 8 wherein the disease is schizophrenia. 